The primary goal of this proposal is to define more precisely the activities of proteins that mediate cell cycle control through the use of the adenovirus E1A protein. E1A is a multifunctional phosphoprotein. Its versatility is reflected in the ability to induce cellular DNA synthesis, overcome the growth inhibitory effect of TGF-beta in epithelial cells, and override the requirement for cellular ras activity in promoting cells into S phase. These activities of E1A in overriding the requirement for ras activity. Experiments have been proposed to determine which, if any, of the E1A-associated proteins are required by E1A to induce DNA synthesis in cells that have lost ras activity. We hypothesize that the identities of these proteins will be important since it will establish, for the first time, whether growth suppressors such as pRb, p107 and/or p130 are downstream targets of ras. The second aim of this proposal is to determine the mechanisms by which E1A can overcome the growth-inhibitory effects of TGF-beta in epithelial cells. We hypothesize that E1A can restore kinase activity to cdks by disabling the inhibitory effects of p27Kipl in TGF- beta-treated cells, and present two experimental models in which this may occur. Another aim of this proposal is to identify the cellular proteins that are required by E1A to induce cellular DNA synthesis in quiescent cells. Mutant E1A proteins that fail to interact with a specific set of cellular proteins will be created and tested by microinjection for their ability to initiate DNA synthesis. Also, antibodies or anti-sense oligonucleotides that can neutralize the activities of cyclindependent kinases and other proteins important to the G1 to S transition will be used as tools, to determine whether any of these proteins are needed by E1A to stimulate DNA synthesis. Finally, since E1A is a phosphoprotein, the hypothesis that phosphorylation may have regulatory importance in some of E1A's activities remains a possibility. Thus, E1A mutants impaired in phosphorylation will also be used in some of the proposed studies.